Transcriptional regulation of enteroendocrine cell differentiation by NeuroD

描述(申请人提供)：哺乳动物的肠道不断自我更新，因为肠道干细胞产生四种上皮细胞类型。肠内分泌细胞不到上皮细胞总数的5%，但分泌的激素控制着许多生理过程，包括食欲和饱腹感、胰岛素分泌和消化器官功能。转录因子神经原3在肠道中启动内分泌分化程序，并激活另一种基本螺旋环状螺旋蛋白Neurd的表达。Neurd似乎协调了肠内分泌细胞的终末分化和细胞周期退出。神经原蛋白3表达细胞中Wnt信号的激活可诱导肠神经内分泌肿瘤的发生，而神经原蛋白3表达细胞中Wnt信号的激活不能诱导肠神经内分泌肿瘤的发生，提示神经原蛋白3的表达代表了肠内分泌细胞分化的一个明显的后期阶段。NeuroD的转录激活机制尚不清楚，但初步结果表明，它与其他DNA结合蛋白CtBP以及组蛋白修饰酶赖氨酸特异性去甲基酶1(LSD1)相互作用。肠内分泌细胞中识别的神经靶点的缺乏，使得人们很难理解这种重要的转录因子在其分化中的作用。这项提案的三个目标将解决神经D在分化肠内分泌细胞方面的功能。目标1将研究神经D如何与Sp1、RREB1和LSD1在其已知靶点之一--促胰液素基因--形成多蛋白共激活复合体。C端结合蛋白CtBP通常是一种辅阻遏子，其矛盾的共激活功能将通过检测组蛋白修饰以及与分泌素基因增强子CtBP和Neurd相关的蛋白来表征。目标2的目标是通过在体外测定神经D与Wnt/β-catenin调控的AT启动子相关的DNA结合蛋白和辅助激活/辅助抑制物复合体，来研究神经D抑制Wnt信号转导的转录机制。在体内，将在有条件地表达Neurod或Neurod敲除shRNA的转基因小鼠中检测NeuD在抑制Wnt信号转导中的作用，以确定NeuroD在分化早期的表达是否阻止了Wnt激活后神经内分泌肿瘤的发展，或者敲除Neurod表达是否解除了对NeuroD+细胞中Wnt激活后肿瘤发展的阻碍。目的利用本课题组开发的一种新方法，通过高通量测序(RNAseq)从小鼠小肠中分离的神经D+细胞的RNA进行基因表达谱分析，从而鉴定正常肠内分泌细胞中神经调节基因。全基因组染色质占有率研究(ChIPseq)将确定差异表达基因的子集作为神经D的潜在直接靶点。

DESCRIPTION (provided by applicant): The mammalian intestine continuously renews itself as intestinal stem cells give rise to four epithelial cell types. Enteroendocrine cells represent less than 5% of the total number of epithelial cells but secrete hormones that control numerous physiological processes including appetite and satiety, insulin secretion, and digestive organ function. The transcription factor, Neurogenin 3, initiates the endocrine differentiation program in the intestine and activates expression of NeuroD, another basic helix loop helix protein. NeuroD appears to coordinate terminal differentiation of enteroendocrine cells with cell cycle exit. Activation of constitutive Wnt signaling in neurogenin 3 expressing cells induced intestinal neuroendocrine tumors whereas Wnt activation in NeuroD expressing cells did not, suggesting that NeuroD expression represents a distinct, later stage of differentiation of enteroendocrine cells. The mechanism of transcriptional activation by NeuroD is not well characterized but preliminary results indicate interactions with other DNA binding proteins, CtBP, and the histone modifying enzyme, lysine specific demethylase 1 (LSD1) are involved. The paucity of identified NeuroD targets in enteroendocrine cells, has made it difficult to understand the role of this important transcription factor in their differentiation. The three aims of this proposal will address the function of NeuroD in differentiating enteroendocrine cells. Aim 1 will examine how NeuroD associates with Sp1, RREB1, and LSD1 at one its known targets, the secretin gene to form a multiprotein coactivator complex. The paradoxical coactivator function of C-terminal binding protein, CtBP, which is generally a corepressor, will be characterized by examining histone modifications and proteins CtBP and NeuroD associate with at the secretin gene enhancer. The goal of Aim 2 will study the transcriptional mechanism of inhibition of Wnt signaling by NeuroD by determining the DNA binding proteins and coactivators/corepressor complexes that NeuroD associates with at promoters regulated by Wnt/?-catenin in vitro. The role of NeuroD in the inhibition of Wnt signaling in vivo will be examined in transgenic mice that either conditionally express NeuroD or a NeuroD knockdown shRNA to determine whether expression of NeuroD at an earlier stage of differentiation prevents development of neuroendocrine tumors following Wnt activation or whether knocking down NeuroD expression removes the block to developing tumors following Wnt activation in NeuroD+ cells. Aim 3 will identify NeuroD regulated genes in normal enteroendocrine cells by gene expression profiling by high throughput sequencing (RNAseq) of RNA from NeuroD+ cells isolated from mouse small intestine by a new method developed by the principal investigator. Genome-wide chromatin occupancy studies (ChIPseq) will identify a subset of differentially expressed genes as potential direct targets by NeuroD.